I have a number of issues post-build trying to focus on my first test slide. I’m listing them in case they are related to each other.
- I cannot get the slide to focus. What I see on the screen is totally out of focus, but the color is white, then light green when I insert the slide, so I know the slide object (honey bee part) is centered. If I disconnect the Z stepper motor and move through the entire gear range, it never comes even remotely into focus. I do see a moment of focus on what appears to be perhaps dust on glass, and if I remove the slide the same dots appear, background is now white, so I think I am focusing on the objective lens glass itself, not the slide object.
- When I try auto-calibrate, I hear the stepper motors, initially short buzz bursts, eventually longer bursts, but the gears do not turn, at least that I can see. I removed X and Z motors, the gears seem to move freely by hand. The X motor is quite hot to the touch, Z is slightly warm, Y is cold.
I bought all my parts from IO Rodeo, including the objective, Sangaboard and PI 4B, the motors are 5V, so I assume the parts are correct. One issue I did have during build was that the objective extended up onto the slide deck, so I couldn’t get a slide into the clips. Rather than push the objective to the top of its slot like the instructions say, I had to lower it before tightening the screw such that the objective just clears the bottom of the slide.
Hoping for suggestions,
Thanks, Kevan
Welcome to the Forum @kevan.rehm. What optics module are you using? From what you have said it seems that you are using an RMS objective, what magnification? You say that is also from IO Rodeo?
A photograph of the microscope with the slide in place might help to diagnose the issues
Objective says: Plan 40/0.65. 160/0.17
This shows the problem. The working distance of a x40 lens is much less than 1mm. They cannot focus through a microscope slide, only through the thin coverslip that covers the sample.
Turn the slide over, so that the sample is on the same side as the lens. Then you should get focus when the objective is nearly, but not quite, touching the coverslip.
1 Like
I reversed the slide so that the slip was down. Also moved the objective slightly closer to the slide, there is not much more than a piece of paper thickness distance between objective and slip cover, still no improvement in focus.
This could be a red herring, but what would the symptoms be if the condenser lens would be put in upside down? I ask because I had difficulty determining which was the flat side and which was the curved side. I thought I got it right, but if this would explain the symptoms, then perhaps I need to do that part over. Otherwise I can’t think of what could be wrong.
When I run the autocalibrate with a specimen in the center, I can by see by color movement that the stage moves around before it gives up, moved -32768 with no object seen. If I select move to 0:0:0 it moves back to the original spot, so that seems. okay.
Autocalibration of the stage motion will not work until you have got the image in focus, it needs to see something clearly move and measure the shift. Your problem is focus.
The condenser lens may be the issue, or do you mean the tube lens in the optics module, between the objective and the camera?
The condenser lens in the illumination above the stage does not itself affect the focus, but if that lens was flat, it is not the correct lens. The tube lens is almost flat on both sides, the lens in the illumination is almost a hemisphere on one side. They are both similar diameter. If they are swapped one for the other it would make a big difference.
IO Rodeo did a good job of labeling each part in a separate plastic bag, so I don’t think I have the lenses reversed.
The reflection seems to indicate that the condenser lens is semi-circular. So the only issue I can think of is that the lens in the optics module is backwards (that’s what I actually meant when I said I had problems picking the orientation). Do my symptoms make sense for a backwards optics lens? I don’t see a way to get the lens out of the optics module. (Photo of the optics module lens coming next.)
This is what the screen looks like, during “Auto-calibrate using camera”, I have the lens actually touching the slip cover, and I see no change in focus during the test.
There will be no change in focus during any of the calibration.
I am at a bit of a loss as to what the problem is here. The tube lens being upside-down would make a small change in focal position, but not enough I think. You have already tried the whole range of motion by hand, taking the motor off. Have you done that after you got yhe slide the correct way up?
The other test is if you have a spare blank slide, draw on it with a permanent marker, and put that marker side down on the microscope. The streaky pen gives a very good flat object to image usually.
With your bee slide try positioning it so that the edge of the coverslip is in the middle of the field of view, all being well that gives an out of focus dark line across the image. You could also try the edge of the label. The big high contrast edge often gives a clearer indication of when you are heading towards focus, even when you are far away. Is it that you cannot get close enough to focus, or you cannot get far enough away?
So, progress finally. I removed the 4 bolts holding the stage in the base, then I lifted the stage out of the base. I loosened the set screw on the optics module, then slowly moved the module up and down while watching the monitor until I saw the slide image somewhat in focus, then tightened the screw. If I set the Z axis to -5000, I can get the image to focus cleanly. So there was nothing wrong with the hardware or the build, just getting the optics module within a range that could display the image. It is really touchy to get this right; I tried positioning it closer to Z axis zero, but no luck, slight hand movements result in wild swings on the screen, not only up and down but also left and right, forward and backward, so I’ve left it where it is.
I wonder if there is a way to incorporate a thumbwheel that would move the optics module up and down in minute increments, and limiting left/right/forward/backward motion so that the image was more stable while positioning the module, making it easier to get closer to Z zero, and keeping the lens centered in the middle of the stage. Just a suggestion, seems like a feature that would be really helpful. I don’t (yet) have the 3D printing skills to do this.
